Trinh Thi B, Pei Dehua
Department of Chemistry and Biochemistry, The Ohio State University, 578 Biosciences Building, 484 West 12th Avenue, Columbus, OH, 43210, USA.
Methods Mol Biol. 2016;1360:169-81. doi: 10.1007/978-1-4939-3073-9_13.
Protein kinases phosphorylate specific serine, threonine, and/or tyrosine residues in their target proteins, resulting in functional changes of the target proteins such as enzymatic activity, cellular location, or association with other proteins. For many kinases, their in vivo substrate specificity is at least partially defined by the amino acid sequence surrounding the phosphorylatable residue (or sequence specificity). We report here a robust, high-throughput method for profiling the sequence specificity of protein kinases. Up to 10(7) different peptides are rapidly synthesized on PEGA beads in the one-bead-one-compound format and subjected to kinase reaction in the presence of [γ-S]ATP. Positive beads displaying the optimal kinase substrates are identified by covalently labeling the thiophosphorylated peptides with a fluorescent dye via a disulfide exchange reaction. Finally, the most active hit(s) is identified by the partial Edman degradation-mass spectrometry (PED-MS) method. The ability of this method to provide individual sequences of the preferred substrates permits the identification of sequence contextual effects and non-permissive residues. This method is applicable to protein serine, threonine, and tyrosine kinases.
蛋白激酶使其靶蛋白中的特定丝氨酸、苏氨酸和/或酪氨酸残基磷酸化，从而导致靶蛋白的功能发生变化，如酶活性、细胞定位或与其他蛋白的结合。对于许多激酶而言，其体内底物特异性至少部分由可磷酸化残基周围的氨基酸序列（即序列特异性）所决定。我们在此报告一种用于分析蛋白激酶序列特异性的强大的高通量方法。以单珠单化合物形式在聚乙二醇胺（PEGA）珠上快速合成多达10⁷种不同的肽，并在[γ-S]ATP存在的情况下进行激酶反应。通过二硫键交换反应，用荧光染料共价标记硫代磷酸化肽，从而鉴定出显示最佳激酶底物的阳性珠。最后，通过部分埃德曼降解-质谱（PED-MS）方法鉴定出活性最高的命中肽段。该方法提供优选底物的单个序列的能力允许鉴定序列上下文效应和非允许残基。此方法适用于蛋白丝氨酸、苏氨酸和酪氨酸激酶。
